Vaccine against RSV

ABSTRACT

The present invention relates to novel nucleic acid molecules encoding a pre-fusion RSV F protein or immunologically active part thereof, wherein the pre-fusion RSV F protein comprises the amino acid sequence of SEQ ID NO: 1 or 2. The invention further relates to the use of the nucleic acid molecules, or vectors comprising said nucleic acid molecules, as a vaccine against respiratory syncytial virus (RSV).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/946,485 filed Jun. 24, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/091,257, filed Oct. 4, 2018, which is a Section371 of International Application No. PCT/EP2017/057957, filed Apr. 4,2017, which was published in the English language on Oct. 12, 2017,under International Publication No. WO 2017/174564 A1, which claimspriority under 35 U.S.C. § 119(b) to European Application No.16163807.7, filed Apr. 5, 2016, the disclosures of which areincorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “Sequence_Listing_004852-111US3”, creation date of May 12,2022, and having a size of about 16 KB. The sequence listing submittedvia EFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of medicine. More in particular, theinvention relates to vaccines against RSV.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is a highly contagious childhoodpathogen of the respiratory tract which is believed to be responsiblefor 200,000 childhood deaths annually. In children younger than 2 years,RSV accounts for approximately 50% of the hospitalizations due torespiratory infections, with a peak of hospitalization occurring at 2-4months of age. It has been reported that almost all children will haveexperienced infection with RSV by the age of two, and repeated infectionduring life is attributed to low natural immunity. In the elderly, theRSV disease burden is similar to those caused by non-pandemic influenzaA infections. A vaccine against RSV is currently not available, but isdesired due to the high disease burden.

RSV is a paramyxovirus, belonging to the subfamily of pneumovirinae. Itsgenome encodes for various proteins, including the membrane proteinsknown as RSV Glycoprotein (G) and RSV fusion (F) protein which are themajor antigenic targets for neutralizing antibodies.

Unlike the RSV G protein, the F protein is conserved between RSVstrains; which makes it an attractive vaccine candidate able to elicitbroadly neutralizing antibodies. The F protein is a transmembraneprotein and it is incorporated in the virion membrane from cellularmembrane during virus budding. The RSV F protein facilitates infectionby fusing the viral and host-cell membranes. In the process of fusion,the F protein refolds irreversibly from a labile pre-fusion conformationto a stable post-fusion conformation. The protein precursor, F0,requires cleavage during intracellular maturation by a furin-likeprotease. There are two furin sites, cleavage of which results inremoval of a p27 peptide and formation of two domains: an N-terminal F2domain and a C-terminal F1 domain (FIG. 1 ). The F2 and F1 domains areconnected by two cystine bridges. Antibodies against the fusion proteincan prevent virus uptake in the cell and thus have a neutralizingeffect. Besides being a target for neutralizing antibodies, RSV Fcontains cytotoxic T cell epitopes (Pemberton et al, 1987, J. Gen.Virol. 68: 2177-2182).

Despite 50 years of research, there is still no licensed vaccine againstRSV. One major obstacle to the vaccine development is the legacy ofvaccine-enhanced disease in a clinical trial in the 1960s with aformalin-inactivated (FI) RSV vaccine. FI-RSV vaccinated children werenot protected against natural infection and infected childrenexperienced more severe illness than non-vaccinated children, includingtwo deaths. This phenomenon is referred to as ‘enhanced disease’.

Since the trial with the FI-RSV vaccine, various approaches to generatean RSV vaccine have been pursued. Attempts include classical liveattenuated cold passaged or temperature sensitive mutant strains of RSV,(chimeric) protein subunit vaccines, peptide vaccines and RSV proteinsexpressed from recombinant viral vectors, including adenoviral vectors.Although some of these vaccines showed promising pre-clinical data, novaccine has been licensed for human use due to safety concerns or lackof efficacy.

Therefore, a need remains for efficient vaccines and methods ofvaccinating against RSV, in particular vaccines that do not lead toenhanced disease. The present invention aims at providing such vaccinesand methods for vaccinating against RSV in a safe and efficaciousmanner.

SUMMARY OF THE INVENTION

The present invention provides novel nucleic acid molecules encodingstable RSV pre-fusion F proteins, wherein the RSV pre-fusion F proteinscomprise the amino acid sequence of SEQ ID NO: 1 or 2.

In certain embodiments, the nucleic acid molecules encoding the RSVpre-fusion F proteins are codon optimized for expression in human cells.

In certain embodiments, the nucleic acid molecules encoding the RSVpre-fusion F proteins comprise the nucleic acid sequence of SEQ ID NO: 3or 4.

The invention further provides vectors comprising the nucleic acidmolecules encoding RSV pre-fusion F proteins, wherein the RSV F proteincomprises the amino acid sequence of SEQ ID NO: 1 or 2.

In certain embodiments, the vector is a human recombinant adenovirus.

In certain embodiments, the recombinant adenovirus is of serotype 26 or35.

In certain embodiments, the recombinant human adenovirus has a deletionin the E1 region, a deletion in the E3 region, or a deletion in both theE1 and the E3 region of the adenoviral genome.

In certain embodiments, the recombinant adenovirus has a genomecomprising at its 5′ terminal ends the sequence CTATCTAT.

The invention also provides compositions, e.g. vaccines againstrespiratory syncytial virus (RSV), comprising a nucleic acid molecule ora vector according to the invention.

The invention further provides a method for vaccinating a subjectagainst RSV, the method comprising administering to the subject acomposition according to the invention.

In certain embodiments, the method of vaccinating a subject against RSVfurther comprises administering RSV F protein (preferably formulated asa pharmaceutical composition, thus a protein vaccine) to the subject.

The invention also provides a method for reducing infection and/orreplication of RSV in, e.g. the nasal tract and lungs of, a subject,comprising administering to the subject a composition comprising anucleic acid or vector according to the invention. This will reduceadverse effects resulting from RSV infection in a subject, and thuscontribute to protection of the subject against such adverse effectsupon administration of the composition. In certain embodiments, adverseeffects of RSV infection may be essentially prevented, i.e. reduced tosuch low levels that they are not clinically relevant.

The invention also provides an isolated host cell comprising a nucleicacid molecule encoding a RSV pre-fusion F protein, wherein the RSVpre-fusion F protein comprises the amino acid sequence of SEQ ID NO: 1or 2. In certain embodiments, the nucleic acid molecule encoding the RSVpre-fusion F protein comprises the nucleic acid sequence of SEQ ID NO: 3or 4.

The invention further provides a method for making a vaccine againstrespiratory syncytial virus (RSV), comprising providing a recombinanthuman adenovirus that comprises nucleic acid encoding a RSV pre-fusion Fprotein or fragment thereof, propagating said recombinant adenovirus ina culture of host cells, isolating and purifying the recombinantadenovirus, and formulating the recombinant adenovirus in apharmaceutically acceptable composition, wherein the RSV pre-fusion Fprotein comprises the amino acid sequence of SEQ ID NO: 1 or 2. Incertain embodiments, the recombinant adenovirus is of serotype 26 or 35.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Schematic representation of the RSV F protein. A proteinprecursor includes F2 and F1 domains and p27 peptide that is removedfrom the mature proteins by cleavage with furin-like proteases. Thecleavage sites are indicated by arrows. The numbers on top of the boxesindicate amino acid positions in the full length protein excludingsignal peptide. In the F1, structural elements are shown: fusion peptide(FP), refolding region 1 (RR1) including heptad repeat A (HRA) andrefolding region 2 (RR2) including heptad repeat B (HRB).

FIG. 2 : Relative surface expression of F protein variants. Full lengthvariants of F protein were expressed in HEK293T cells. The cell werestained with anti-RSV F antibody (CR9503) and analyzed by Flow Cytometry(FACS). The Mean Fluorescent Intensity (MFI) values were calculated andnormalized to MFI of control F wild type (Fwt)-transfected cell sample.The MFI of Fwt was set to 1. Bars represent mean values, error barsrepresent range of values.

FIG. 3 : Fraction of pre-fusion F protein on cell surface. Full lengthvariants of F protein were expressed in HEK293T cells. The cell werestained with anti-RSV F antibody CR9503 and anti-pre-fusion RSV Fantibody CR9501 and analyzed by Flow Cytometry. The Mean FluorescentIntensity (MFI) values were calculated and MFI measured by CR9501 wasnormalized to MFI measured by CR9503. The normalized MFI values indicatefraction of the pre-F on the cell surface. Bars represent mean values,error bars represent range of values.

FIG. 4 : Relative surface expression of F protein variants. Solubleversions with the transmembrane region and cytoplasmic region deleted(Fsl) and full length variants of F protein were expressed in HEK293Tcells. The expression level of the soluble protein was measured in theculture supernatant by octet and the full length variants were tested bycell staining using anti-RSV F antibody (CR9503) and analyzed by FlowCytometry. The Mean Fluorescent Intensity (MFI) values were calculatedand normalized to MFI of control F wild type (Fwt)-transfected cellsample. The MFI of Fwt was set to 1. Bars represent mean values, errorbars represent range of values.

FIGS. 5A and 5B: Temperature stability of the F protein variants. Fulllength variants of F protein were expressed in HEK293T cells. Afterheat-shock, the cell were stained with anti-RSV F antibody (CR9501—solidlines and CR9503—dashed lines) and analyzed by Flow Cytometry. Thepercentage of cells positive for the staining was determined. Symbolsrepresent mean values, error bars represent range of values. (FIG. 5A)The percentage of cells positive for the staining was determined. (FIG.5B) The Mean Fluorescent Intensity (MFI) values were calculated andnormalized to MFI of 37 C cell sample. The MFI of 37° C. sample was setto 1. Dotted lines correspond to background staining at 60° C.

FIG. 6 : Stability of the F proteins. PreF is more stable than FA2protein in prefusion conformation on the cell surface in the heat-stressassay. A549 cells were infected with Ad26 and Ad35 comprising the insertFA2 (wt RSV F, grey bars) or prefusion F stabilized insert (preF2.2,black bars) at the indicated MOI. The cells were temperature treated atthe indicated temperature for 15 minutes before staining. Top:percentage of cells presenting prefusion F on their surface (detected byCR9501 antibody); bottom: percentage of cells presenting any form of theF protein, prefusion and post fusion (detected by CR9503 antibody). Thevalues were normalized to 37° C. samples. All bars represent a singlemeasurement.

FIG. 7 : Ad26.RSV.preF2.1 and F2.2 elicit a cellular immune responseafter single administration in mice. Horizontal bars depict thegeometric mean of the response within a group. The background level iscalculated as the 95% percentile of the spot forming units (SFU)observed in non-stimulated splenocytes, and is indicated with a dottedline.

FIGS. 8A, 8B, and 8C: Ad26.RSV.preF2.1 and F2.2 induce increased virusneutralizing antibodies when compared to Ad26.RSV.FA2, after singleimmunization in mice. Balb/c mice (n=4 per group) were immunized withthe indicated dose of 108 to 1010 viral particles (vp) Ad26.RSV.FA2 orAd26.RSV.preF2.1 or Ad26.RSV.preF2.2, or with formulation buffer, andhumoral immune responses were assayed in the serum isolated 8 weeksafter immunization. (FIG. 8A) Virus neutralizing antibodies weredetermined against RSV A Long using a micro-neutralization assay with anELISA based read out. Titers are given as the log 2 value of the IC50.(FIG. 8B) Pre-fusion or post-fusion F antibody titers were determined byELISA, and the ratio between pre- and post-fusion F antibodies for allsamples that showed pre- and post-fusion F titers above lower limit ofquantification (LLoQ) was calculated. (FIG. 8C) Subclass ELISA wasperformed using post-fusion RSV F A2 as coating reagent, and theIgG2a/IgG1 ratio (log 10) is plotted. Ratio's observed for Th1 (serumderived from animals immunized with RSV F expression Adenoviral vectors)and Th2 (serum derived from FI-RSV immunized animals) references samplesare indicated with dashed lines. The LLoQ is indicated with a dottedlines (panels A), and horizontal bars represent the mean responses pergroup.

FIG. 9 : Ad26.RSV.preF2.2 elicits antibody responses that neutralize awide range of RSV isolates. Sera from Balb/c mice that were immunizedwith 1010 viral particles (vp) Ad26.RSV.FA2 (n=3) or Ad26.RSV.preF (n=4)or formulation buffer (n=2) were used in virus neutralization assays(micro-neutralization assay with an ELISA based read out) with the RSV A(upper panels) and B strains (lower panels) indicated. Titers are givenas the log 2 value of the IC50, and horizontal bars represent the meanresponse per group. LLoQ is indicated with a dashed line.

FIGS. 10A, 10B, 10C, 10D, 10E, and 10F: Single immunization withAd26.RSV.preF2.2 or Ad35.RSV.preF2.2 at low doses protects cotton ratsagainst challenge with the homologous RSV A2. Cotton rats (Sigmodonhispidus) (n=7 to 9 per group) were immunized with the indicated doses(in vp/animal) of Ad26.RSV.preF2.2, Ad26.RSV.FA2 (left panels),Ad35.RSV.preF2.2 or Ad35.RSV.FA2 (right panels) by single intramuscularadministration. Control immunizations were performed with formulationbuffer, FI-RSV, or intranasal application of a low dose of RSV A2. Atseven weeks post-immunization animals were challenged intranasally with105 pfu RSV A2. The lung (FIGS. 10A and 10B) and nose viral titers(FIGS. 10C and 10D) were determined by plaque assay 5 days afterchallenge. (FIGS. 10E and 10F) Sera taken just before challenge wereused to perform a virus neutralizing assay with the RSV A Long strain(micro-neutralization assay with an ELISA based read out). The dottedline represents the lower level of quantification (LLoQ). Horizontalbars represent the mean titer per group.

FIG. 11 : Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2 immunization of cottonrats does not result in increased alveolitis scores after RSV A2challenge. Cotton rats (Sigmodon hispidus) (n=7 to 9 per group) wereimmunized with the indicated doses (in vp/animal) of Ad26.RSV.preF2.2,Ad26.RSV.FA2 (upper panel), Ad35.RSV.preF2.2, or Ad35.RSV.FA2 (lowerpanel) by single intramuscular administration. Control immunizationswere performed with formulation buffer, FI-RSV, or intranasalapplication of a low dose of RSV A2. At seven weeks post-immunizationanimals were challenged intranasally with 105 pfu RSV A2. Alveolitis wasscored by histopathological examination of one lung lobe 5 days afterchallenge on a non-linear scale from 0 to 4. The horizontal dotted linemarks the maximal score of the control animals that were pre-exposed toRSV-A2 before challenge to mimic a natural exposure to RSV that does notlead to ERD.

DETAILED DESCRIPTION OF THE INVENTION

RSV vaccines comprising human adenovirus comprising nucleic acidencoding RSV F protein have previously been described in WO2013/139911and WO2013/139916. It was shown therein that recombinant adenoviruses ofserotype 26 or 35 that comprise a nucleic acid encoding an RSV F proteinare very effective vaccines against RSV in a well established cotton ratmodel and have improved efficacy as compared to data described earlierfor Ad5 encoding RSV F protein. It was demonstrated that a singleadministration, even administered intramuscularly, of Ad26 or Ad35encoding RSV F is sufficient to provide complete protection againstchallenge RSV replication.

Because of the instability of the RSV F protein, however, the RSV Fprotein has the propensity to prematurely refold into its more stablepost-fusion conformation. This phenomenon is an intrinsic feature of theprotein both in solution and on the surface of the virions. In humansera most RSV neutralizing antibodies are, however, directed against theRSV F in the pre-fusion conformation. In the research that led to thepresent invention, effort was therefore undertaken to identifymodifications that stabilize RSV F protein in its pre-fusionconformation in the full length protein.

Several possible mutations stabilizing RSV F protein in the pre-fusionconformation have previously been described in WO2014/174018 andWO2014/202570. The nucleic acid molecules according to the presentinvention encode RSV F proteins comprising a unique and specific subsetof mutations. According to the invention it has been shown that thisunique combination of mutations of the present invention results inincreased RSV F protein expression levels and stability of the RSV Fprotein in the pre-fusion conformation. In addition, it has been shownthat the nucleic acid molecules according to the invention encode RSV Fprotein which is stabilized in the pre-fusion conformation, and whichinduces higher titers of neutralizing antibody as compared to wild typeRSV F protein (as disclosed in WO2013/139911 and WO2013/139916).

In a first aspect, the present invention thus provides novel nucleicacid molecules encoding a pre-fusion respiratory syncytial virus Fprotein (RSV pre-fusion F protein, or RSV pre-F protein), wherein theRSV pre-F protein comprises the amino acid sequence of SEQ ID NO: 1 or2.

In certain embodiments, the nucleic acid molecules encode the RSV pre-Fproteins of SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the terms nucleic acid, nucleic acid molecule, nucleicacid or nucleotide sequence, and polynucleotide are used interchangeablyand all refer to the linear biopolymers (chains) made from nucleotides,including DNA and RNA.

It is understood by a skilled person that numerous different nucleicacid molecules can encode the same polypeptide as a result of thedegeneracy of the genetic code. It is also understood that skilledpersons may, using routine techniques, make nucleotide substitutionsthat do not affect the polypeptide sequence encoded by thepolynucleotides described there to reflect the codon usage of anyparticular host organism in which the polypeptides are to be expressed.Therefore, unless otherwise specified, a “nucleic acid molecule encodingan amino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons. Sequences herein are provided from 5′ to 3′ direction, ascustom in the art.

In certain embodiments, the nucleic acid molecule encodes a fragment ofpre-fusion RSV F comprising the amino acid sequence of SEQ ID NO: 1 or2.

In certain embodiments, the nucleic acid molecules encoding the RSVpre-fusion F protein, or fragment thereof, are codon optimized forexpression in mammalian cells, such as human cells. Methods ofcodon-optimization are known and have been described previously (e.g. WO96/09378).

In certain embodiments, the nucleic acid molecule encoding the RSVpre-fusion F protein comprises the nucleic acid sequence of SEQ ID NO:3. In certain embodiments, the nucleic acid encoding the RSV F proteincomprises the nucleic acid sequence of SEQ ID NO: 4.

In certain embodiments, the nucleic acid encoding the RSV F proteinconsists of the nucleic acid sequence of SEQ ID NO: 3 or 4.

The term “fragment” as used herein refers to a peptide that has anamino-terminal and/or carboxy-terminal and/or internal deletion, butwhere the remaining amino acid sequence is identical to thecorresponding positions in the sequence of the full length RSV Fprotein, for example, the RSV F protein of SEQ ID NO. 1 or 2. It will beappreciated that for inducing an immune response and in general forvaccination purposes, a protein needs not to be full length nor have allits wild type functions, and fragments of the protein are equallyuseful. The person skilled in the art will also appreciate that changescan be made to a protein, e.g. by amino acid substitutions, deletions,additions, etc, e.g. using routine molecular biology procedures.Generally, conservative amino acid substitutions may be applied withoutloss of function or immunogenicity of a polypeptide. This can easily bechecked according to routine procedures well known to the skilledperson.

The present invention also relates to vectors comprising a nucleic acidmolecule encoding a pre-fusion RSV F protein comprising the amino acidsequence of SEQ ID NO: 1 or 2, or a fragment thereof.

According to the invention, the vector may be any vector that can beconveniently subjected to recombinant DNA procedures and can bring aboutexpression of the nucleic acid molecule of the invention. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced.

In certain embodiments, the vector is a human recombinant adenovirus,also referred to as recombinant adenoviral vectors. The preparation ofrecombinant adenoviral vectors is well known in the art. The term‘recombinant’ for an adenovirus, as used herein implicates that it hasbeen modified by the hand of man, e.g. it has altered terminal endsactively cloned therein and/or it comprises a heterologous gene, i.e. itis not a naturally occurring wild type adenovirus.

In certain embodiments, an adenoviral vector according to the inventionis deficient in at least one essential gene function of the E1 region,e.g. the Ela region and/or the E1b region, of the adenoviral genome thatis required for viral replication. In certain embodiments, an adenoviralvector according to the invention is deficient in at least part of thenon-essential E3 region. In certain embodiments, the vector is deficientin at least one essential gene function of the E1 region and at leastpart of the non-essential E3 region. The adenoviral vector can be“multiply deficient,” meaning that the adenoviral vector is deficient inone or more essential gene functions in each of two or more regions ofthe adenoviral genome. For example, the aforementioned E1-deficient orE1-, E3-deficient adenoviral vectors can be further deficient in atleast one essential gene of the E4 region and/or at least one essentialgene of the E2 region (e.g., the E2A region and/or E2B region).

Adenoviral vectors, methods for construction thereof and methods forpropagating thereof, are well known in the art and are described in, forexample, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806,5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and6,113,913, and Thomas Shenk, “Adenoviridae and their Replication”, M. S.Horwitz, “Adenoviruses”, Chapters 67 and 68, respectively, in Virology,B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996),and other references mentioned herein. Typically, construction ofadenoviral vectors involves the use of standard molecular biologicaltechniques, such as those described in, for example, Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA,2d ed., Scientific American Books (1992), and Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience Publishers, NY(1995), and other references mentioned herein.

In certain embodiments, the adenovirus is a human adenovirus of theserotype 26 or 35. The vaccines according to the invention based onthese serotypes appear more potent than the ones described in the priorart that were based on Ad5, since those failed to provide completeprotection against RSV challenge replication after a singleintramuscular administration (Kim et al, 2010, Vaccine 28: 3801-3808;Kohlmann et al, 2009, J Virol 83: 12601-12610; Krause et al, 2011,Virology Journal 8:375). The serotype of the invention further generallyhas a low seroprevalence and/or low pre-existing neutralizing antibodytiters in the human population. Recombinant adenoviral vectors of theseserotypes with different transgenes are evaluated in clinical trials,and thus far shows to have an excellent safety profile. Preparation ofrAd26 vectors is described, for example, in WO 2007/104792 and in Abbinket al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26are found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO2007/104792. Preparation of rAd35 vectors is described, for example, inU.S. Pat. No. 7,270,811, in WO 00/70071, and in Vogels et al., (2003) JVirol 77(15): 8263-71. Exemplary genome sequences of Ad35 are found inGenBank Accession AC_000019 and in FIG. 6 of WO 00/70071.

A recombinant adenovirus according to the invention may bereplication-competent or replication-deficient. In certain embodiments,the adenovirus is replication deficient, e.g. because it contains adeletion in the E1 region of the genome. As known to the skilled person,in case of deletions of essential regions from the adenovirus genome,the functions encoded by these regions have to be provided in trans,preferably by the producer cell, i.e. when parts or whole of E1, E2and/or E4 regions are deleted from the adenovirus, these have to bepresent in the producer cell, for instance integrated in the genomethereof, or in the form of so-called helper adenovirus or helperplasmids. The adenovirus may also have a deletion in the E3 region,which is dispensable for replication, and hence such a deletion does nothave to be complemented.

A producer cell (sometimes also referred to in the art and herein as‘packaging cell’ or ‘complementing cell’ or ‘host cell’) that can beused can be any producer cell wherein a desired adenovirus can bepropagated. For example, the propagation of recombinant adenovirusvectors is done in producer cells that complement deficiencies in theadenovirus. Such producer cells preferably have in their genome at leastan adenovirus E1 sequence, and thereby are capable of complementingrecombinant adenoviruses with a deletion in the E1 region. AnyE1-complementing producer cell can be used, such as human retina cellsimmortalized by E1, e.g. 911 or PER.C6 cells (see U.S. Pat. No.5,994,128), E1-transformed amniocytes (See EP patent 1230354),E1-transformed A549 cells (see e.g. WO 98/39411, U.S. Pat. No.5,891,690), GH329:HeLa (Gao et al, 2000, Human Gene Therapy 11:213-219), 293, and the like. In certain embodiments, the producer cellsare for instance HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SFcells, and the like.

For non-subgroup C E1-deficient adenoviruses such as Ad35 (subgroup B)or Ad26 (subgroup D), it is preferred to exchange the E4-orf6 codingsequence of these non-subgroup C adenoviruses with the E4-orf6 of anadenovirus of subgroup C such as Ad5. This allows propagation of suchadenoviruses in well known complementing cell lines that express the E1genes of Ad5, such as for example 293 cells or PER.C6 cells (see, e.g.Havenga et al, 2006, J. Gen. Virol. 87: 2135-2143; WO 03/104467,incorporated in its entirety by reference herein). In certainembodiments, an adenovirus that can be used is a human adenovirus ofserotype 35, with a deletion in the E1 region into which the nucleicacid encoding RSV F protein antigen has been cloned, and with an E4 orf6region of Ad5. In certain embodiments, the adenovirus in the vaccinecomposition of the invention is a human adenovirus of serotype 26, witha deletion in the E1 region into which the nucleic acid encoding RSV Fprotein antigen has been cloned, and with an E4 orf6 region of Ad5.

In alternative embodiments, there is no need to place a heterologousE4orf6 region (e.g. of Ad5) in the adenoviral vector, but instead theE1-deficient non-subgroup C vector is propagated in a cell line thatexpresses both E1 and a compatible E4orf6, e.g. the 293-ORF6 cell linethat expresses both E1 and E4orf6 from Ad5 (see e.g. Brough et al, 1996,J Virol 70: 6497-501 describing the generation of the 293-ORF6 cells;Abrahamsen et al, 1997, J Virol 71: 8946-51 and Nan et al, 2003, GeneTherapy 10: 326-36 each describing generation of E1 deleted non-subgroupC adenoviral vectors using such a cell line).

Alternatively, a complementing cell that expresses E1 from the serotypethat is to be propagated can be used (see e.g. WO 00/70071, WO02/40665).

For subgroup B adenoviruses, such as Ad35, having a deletion in the E1region, it is preferred to retain the 3′ end of the E1B 55K open readingframe in the adenovirus, for instance the 166 bp directly upstream ofthe pIX open reading frame or a fragment comprising this such as a 243bp fragment directly upstream of the pIX start codon (marked at the 5′end by a Bsu36I restriction site in the Ad35 genome), since thisincreases the stability of the adenovirus because the promoter of thepIX gene is partly residing in this area (see, e.g. Havenga et al, 2006,J. Gen. Virol. 87: 2135-2143; WO 2004/001032, incorporated by referenceherein).

“Heterologous nucleic acid” (also referred to herein as ‘transgene’) inadenoviruses of the invention is nucleic acid that is not naturallypresent in the adenovirus. It is introduced into the adenovirus forinstance by standard molecular biology techniques. In the presentinvention, the heterologous nucleic acid encodes the RSV pre-F protein(or fragment thereof) comprising the amino acid sequence of SEQ ID NO: 1or 2. It can for instance be cloned into a deleted E1 or E3 region of anadenoviral vector. A transgene is generally operably linked toexpression control sequences. This can for instance be done by placingthe nucleic acid encoding the transgene(s) under the control of apromoter. Further regulatory sequences may be added. Many promoters canbe used for expression of a transgene(s), and are known to the skilledperson. A non-limiting example of a suitable promoter for obtainingexpression in eukaryotic cells is a CMV-promoter (U.S. Pat. No.5,385,839), e.g. the CMV immediate early promoter, for instancecomprising nt. −735 to +95 from the CMV immediate early geneenhancer/promoter. A polyadenylation signal, for example the bovinegrowth hormone polyA signal (U.S. Pat. No. 5,122,458), may be presentbehind the transgene(s).

In certain embodiments, the recombinant adenovectors of the inventioncomprise as the 5′ terminal nucleotides the nucleotide sequence:CTATCTAT. These embodiments are advantageous because such vectorsdisplay improved replication in production processes, resulting inbatches of adenovirus with improved homogeneity, as compared to vectorshaving the original 5′ terminal sequences (generally CATCATCA) (see alsopatent application nos. PCT/EP2013/054846 and U.S. Ser. No. 13/794,318,entitled ‘Batches of recombinant adenovirus with altered terminal ends’filed on 12 Mar. 2012 in the name of Crucell Holland B.V.), incorporatedin its entirety by reference herein. The invention thus also providesbatches of recombinant adenovirus encoding RSV F protein or a partthereof, wherein the RSV F protein comprises the amino acid sequence ofSEQ ID NO: 1 or 2, and wherein essentially all (e.g. at least 90%) ofthe adenoviruses in the batch comprise a genome with terminal nucleotidesequence CTATCTAT.

In certain embodiments, the nucleic acid molecule may encode a fragmentof the pre-fusion F protein of RSV. The fragment may result from eitheror both of amino-terminal and carboxy-terminal deletions. The extent ofdeletion may be determined by a person skilled in the art to, forexample, achieve better yield of the recombinant adenovirus. Thefragment will be chosen to comprise an immunologically active fragmentof the F protein, i.e. a part that will give rise to an immune responsein a subject. This can be easily determined using in silico, in vitroand/or in vivo methods, all routine to the skilled person.

The invention furthermore provides compositions comprising a nucleicacid molecule encoding a pre-fusion RSV F comprising the amino acidsequence of SEQ ID NO: 1 or 2.

Also, the invention provides compositions comprising a vector asdescribed herein.

In certain embodiments, the compositions comprising a nucleic acidmolecule and/or a vector are for use in reducing infection and/orreplication of RSV in a subject. In certain preferred embodiments, thecompositions are for use as a vaccine against RSV. The term “vaccine”refers to an agent or composition containing an active componenteffective to induce a therapeutic degree of immunity in a subjectagainst a certain pathogen or disease. In the present invention, thevaccine comprises an effective amount of a nucleic acid molecule thatencodes an RSV pre-fusion F protein comprising SEQ ID NO: 1 or 2, or anantigenic fragment thereof, which results in an immune response againstthe F protein of RSV. This provides a method of preventing serious lowerrespiratory tract disease leading to hospitalization and the decreasethe frequency of complications such as pneumonia and bronchiolitis dueto RSV infection and replication in a subject. Thus, the invention alsoprovides a method for preventing or reducing serious lower respiratorytract disease, preventing or reducing (e.g. shortening) hospitalization,and/or reducing the frequency and/or severity of pneumonia orbronchiolitis caused by RSV in a subject, comprising administering tothe subject a composition comprising a nucleic acid molecule encoding aRSV pre-F protein or fragment thereof, wherein the RSV F proteincomprises the amino acid sequence of SEQ ID NO: 1 or 2. The term“vaccine” according to the invention implies that it is a pharmaceuticalcomposition, and thus typically includes a pharmaceutically acceptablediluent, carrier or excipient. It may or may not comprise further activeingredients. In certain embodiments it may be a combination vaccine thatfurther comprises other components that induce an immune response, e.g.against other proteins of RSV and/or against other infectious agents.

In certain embodiments the compositions comprising the nucleic acidmolecule or vector further comprise, or are administered together with,one or more adjuvants. Adjuvants are known in the art to furtherincrease the immune response to an applied antigenic determinant, andpharmaceutical compositions comprising adenovirus and suitable adjuvantsare for instance disclosed in WO 2007/110409, incorporated by referenceherein. The terms “adjuvant” and “immune stimulant” are usedinterchangeably herein, and are defined as one or more substances thatcause stimulation of the immune system. In this context, an adjuvant isused to enhance an immune response to the RSV prefusion F proteins ofthe invention. Examples of suitable adjuvants include aluminium saltssuch as aluminium hydroxide and/or aluminium phosphate; oil-emulsioncompositions (or oil-in-water compositions), including squalene-wateremulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations,such as for example QS21 and Immunostimulating Complexes (ISCOMS) (seee.g. U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,WO 2005/002620); bacterial or microbial derivatives, examples of whichare monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motifcontaining oligonucleotides, ADP-ribosylating bacterial toxins ormutants thereof, such as E. coli heat labile enterotoxin LT, choleratoxin CT, and the like. It is also possible to use vector-encodedadjuvant, e.g. by using heterologous nucleic acid that encodes a fusionof the oligomerization domain of C4-binding protein (C4 bp) to theantigen of interest (e.g. Solabomi et al, 2008, Infect Immun 76:3817-23). In certain embodiments the compositions of the inventioncomprise aluminium as an adjuvant, e.g. in the form of aluminiumhydroxide, aluminium phosphate, aluminium potassium phosphate, orcombinations thereof, in concentrations of 0.05-5 mg, e.g. from0.075-1.0 mg, of aluminium content per dose.

In other embodiments, the compositions do not comprise adjuvants.

It is also possible according to the invention to administer furtheractive components, in combination with the compositions, e.g. vaccines,according to the invention. Such further active components may comprisee.g. other RSV antigens or vectors comprising nucleic acid encodingthese. Such vectors again may be non-adenoviral or adenoviral, of whichthe latter can be of any serotype. An example of other RSV antigensincludes RSV F or G protein or immunologically active parts thereof. Forinstance, intranasally applied recombinant replication-deficient Ad5based adenovector rAd/3×G, expressing the soluble core domain of Gglycoprotein (amino acids 130 to 230) was protective in a murine model(Yu et al, 2008, J Virol 82: 2350-2357), and although it was notprotective when applied intramuscularly, it is clear from these datathat RSV G is a suitable antigen for inducing protective responses.Further active components may also comprise non-RSV antigens, e.g. fromother pathogens such as viruses, bacteria, parasites, and the like. Theadministration of further active components may for instance be done byseparate administration or by administering combination products of thevaccines of the invention and the further active components. In certainembodiments, further non-adenoviral antigens (besides RSV.F), may beencoded in the vectors of the invention. In certain embodiments, it maythus be desired to express more than one protein from a singleadenovirus, and in such cases more coding sequences for instance may belinked to form a single transcript from a single expression cassette ormay be present in two separate expression cassettes cloned in differentparts of the adenoviral genome.

The compositions of the invention, e.g. the adenovirus compositions, maybe administered to a subject, e.g. a human subject. The total dose ofthe adenovirus provided to a subject during one administration can bevaried as is known to the skilled practitioner, and is generally between1×107 viral particles (vp) and 1×1012 vp, preferably between 1×108 vpand 1×1011 vp, for instance between 3×108 and 5×1010 vp, for instancebetween 109 and 3×1010 vp.

Administration of the compositions can be performed using standardroutes of administration. Non-limiting embodiments include parenteraladministration, such as by injection e.g. intradermal, intramuscular,etc, or subcutaneous, transcutaneous, or mucosal administration, e.g.intranasal, oral, and the like. Intranasal administration has generallybeen seen as a preferred route for vaccines against RSV. The mostimportant advantage of the live intrasal strategy is the directstimulation of local respiratory tract immunity and the lack ofassociated disease enhancement. The only vaccines under clinicalevaluation for pediatric use at the present time are live intranasalvaccine (Collins and Murphy. Vaccines against human respiratorysyncytial virus). In: Perspectives in Medical Virology 14: RespiratorySyncytial Virus (Ed. Cane, P.), Elsevier, Amsterdam, the Netherlands, pp233-277). Intranasal administration is a suitable preferred routeaccording to the present invention as well. The advantage ofintramuscular administration is that it is simple and well-established,and does not carry the safety concerns for intranasal application ininfants younger than 6 months. In one embodiment a composition isadministered by intramuscular injection, e.g. into the deltoid muscle ofthe arm, or vastus lateralis muscle of the thigh. The skilled personknows the various possibilities to administer a composition, e.g. avaccine in order to induce an immune response to the antigen(s) in thevaccine.

A subject as used herein preferably is a mammal, for instance a rodent,e.g. a mouse, a cotton rat, or a non-human-primate, or a human.Preferably, the subject is a human subject. The subject can be of anyage, e.g. from about 1 month to 100 years old, e.g. from about 2 monthsto about 80 years old, e.g. from about 1 month to about 3 years old,from about 3 years to about 50 years old, from about 50 years to about75 years old, etc.

It is also possible to provide one or more booster administrations ofone or compositions, e.g. the vaccines, of the invention. If a boostingvaccination is performed, typically, such a boosting vaccination will beadministered to the same subject at a moment between one week and oneyear, preferably between two weeks and four months, after administeringthe composition to the subject for the first time (which is in suchcases referred to as ‘priming vaccination’). In alternative boostingregimens, it is also possible to administer different vectors, e.g. oneor more adenoviruses of different serotype, or other vectors such asMVA, or DNA, or protein, to the subject after the priming vaccination.It is for instance possible to administer to the subject a recombinantadenoviral vector comprising a nucleic acid sequence encoding thepre-fusion RSV F protein as a prime, and to boost with a compositioncomprising a RSV F protein. In certain embodiments, the RSV F proteinalso is stabilized in the pre-fusion conformation.

In certain embodiments, the administration comprises a priming and atleast one booster administration. In certain embodiments, thecomposition is administered as a priming composition and/or as aboosting composition in a prime-boost vaccination regimen. In certainembodiments thereof, the priming administration is with a rAd35comprising nucleic acid encoding RSV pre-F protein or a fragment thereof(rAd35-RSV.pre-F′), wherein the RSV F pre-F protein comprises the aminoacid sequence of SEQ ID NO: 1 or 2, and the booster administration iswith a rAd26 comprising nucleic acid encoding RSV F protein(rAd26-RSV.pre-F′), wherein the RSV pre-F protein comprises the aminoacid sequence of SEQ ID NO: 1 or 2. In other embodiments thereof, thepriming administration is with rAd26-RSV.pre-F and the boosteradministration is with rAd35-RSV.pre-F. In other embodiments, both thepriming and booster administration are with rAd26.RSV.pre-F. In certainembodiments, the priming administration is with rAd26-RSV.pre-F orrAd35-RSV.pre-F and the booster administration is with RSV F protein. Inall these embodiments, it is possible to provide further boosteradministrations with the same or other vectors or protein. Embodimentswhere boosting with RSV F protein may be particularly beneficial includee.g. in elder subjects in risk groups (e.g. having COPD or asthma) of 50years or older, or e.g. in healthy subjects of 60 years or older or 65years or older.

In certain embodiments, the administration comprises a singleadministration of a composition according to the invention, withoutfurther (booster) administrations. Such embodiments are advantageous inview of the reduced complexity and costs of a single administrationregimen as compared to a prime-boost regimen. Complete protection isalready observed after single administration of the recombinantadenoviral vectors of the invention without booster administrations inthe cotton rat model in the examples herein.

In a further aspect, the invention provides methods for making a vaccineagainst respiratory syncytial virus (RSV), comprising providing arecombinant human adenovirus that comprises nucleic acid encoding a RSVF protein or fragment thereof, propagating said recombinant adenovirusin a culture of host cells, isolating and purifying the recombinantadenovirus, and bringing the recombinant adenovirus in apharmaceutically acceptable composition, wherein the RSV F proteincomprises the amino acid sequence of SEQ ID NO: 1 or 2.

Recombinant adenovirus can be prepared and propagated in host cells,according to well known methods, which entail cell culture of the hostcells that are infected with the adenovirus. The cell culture can be anytype of cell culture, including adherent cell culture, e.g. cellsattached to the surface of a culture vessel or to microcarriers, as wellas suspension culture.

Most large-scale suspension cultures are operated as batch or fed-batchprocesses because they are the most straightforward to operate and scaleup. Nowadays, continuous processes based on perfusion principles arebecoming more common and are also suitable (see e.g. WO 2010/060719, andWO 2011/098592, both incorporated by reference herein, which describesuitable methods for obtaining and purifying large amounts ofrecombinant adenoviruses).

Producer cells are cultured to increase cell and virus numbers and/orvirus titers. Culturing a cell is done to enable it to metabolize,and/or grow and/or divide and/or produce virus of interest according tothe invention. This can be accomplished by methods as such well known topersons skilled in the art, and includes but is not limited to providingnutrients for the cell, for instance in the appropriate culture media.Suitable culture media are well known to the skilled person and cangenerally be obtained from commercial sources in large quantities, orcustom-made according to standard protocols. Culturing can be done forinstance in dishes, roller bottles or in bioreactors, using batch,fed-batch, continuous systems and the like. Suitable conditions forculturing cells are known (see e.g. Tissue Culture, Academic Press,Kruse and Paterson, editors (1973), and R. I. Freshney, Culture ofanimal cells: A manual of basic technique, fourth edition (Wiley-LissInc., 2000, ISBN 0-471-34889-9).

Typically, the adenovirus will be exposed to the appropriate producercell in a culture, permitting uptake of the virus. Usually, the optimalagitation is between about 50 and 300 rpm, typically about 100-200, e.g.about 150, typical DO is 20-60%, e.g. 40%, the optimal pH is between 6.7and 7.7, the optimal temperature between 30 and 39° C., e.g. 34-37° C.,and the optimal MOI between 5 and 1000, e.g. about 50-300. Typically,adenovirus infects producer cells spontaneously, and bringing theproducer cells into contact with rAd particles is sufficient forinfection of the cells. Generally, an adenovirus seed stock is added tothe culture to initiate infection, and subsequently the adenoviruspropagates in the producer cells. This is all routine for the personskilled in the art.

After infection of an adenovirus, the virus replicates inside the celland is thereby amplified, a process referred to herein as propagation ofadenovirus. Adenovirus infection results finally in the lysis of thecells being infected. The lytic characteristics of adenovirus thereforepermits two different modes of virus production. The first mode isharvesting virus prior to cell lysis, employing external factors to lysethe cells. The second mode is harvesting virus supernatant after(almost) complete cell lysis by the produced virus (see e.g. U.S. Pat.No. 6,485,958, describing the harvesting of adenovirus without lysis ofthe host cells by an external factor). It is preferred to employexternal factors to actively lyse the cells for harvesting theadenovirus.

Methods that can be used for active cell lysis are known to the personskilled in the art, and have for instance been discussed in WO 98/22588,p. 28-35. Useful methods in this respect are for example, freeze-thaw,solid shear, hypertonic and/or hypotonic lysis, liquid shear,sonication, high pressure extrusion, detergent lysis, combinations ofthe above, and the like. In one embodiment of the invention, the cellsare lysed using at least one detergent. Use of a detergent for lysis hasthe advantage that it is an easy method, and that it is easily scalable.

Detergents that can be used, and the way they are employed, aregenerally known to the person skilled in the art. Several examples arefor instance discussed in WO 98/22588, p. 29-33. Detergents can includeanionic, cationic, zwitterionic, and nonionic detergents. Theconcentration of the detergent may be varied, for instance within therange of about 0.1%-5% (w/w). In one embodiment, the detergent used isTriton X-100.

Nuclease may be employed to remove contaminating, i.e. mostly from theproducer cell, nucleic acids. Exemplary nucleases suitable for use inthe present invention include Benzonase®, Pulmozyme®, or any other DNaseand/or RNase commonly used within the art. In preferred embodiments, thenuclease is Benzonase®, which rapidly hydrolyzes nucleic acids byhydrolyzing internal phosphodiester bonds between specific nucleotides,thereby reducing the viscosity of the cell lysate. Benzonase® can becommercially obtained from Merck KGaA (code W214950). The concentrationin which the nuclease is employed is preferably within the range of1-100 units/ml. Alternatively, or in addition to nuclease treatment, itis also possible to selectively precipitate host cell DNA away fromadenovirus preparations during adenovirus purification, using selectiveprecipitating agents such as domiphen bromide (see e.g. U.S. Pat. No.7,326,555; Goerke et al., 2005, Biotechnology and bioengineering, Vol.91: 12-21; WO 2011/045378; WO 2011/045381).

Methods for harvesting adenovirus from cultures of producer cells havebeen extensively described in WO 2005/080556.

In certain embodiments, the harvested adenovirus is further purified.Purification of the adenovirus can be performed in several stepscomprising clarification, ultrafiltration, diafiltration or separationwith chromatography as described in for instance WO 05/080556,incorporated by reference herein. Clarification may be done by afiltration step, removing cell debris and other impurities from the celllysate. Ultrafiltration is used to concentrate the virus solution.Diafiltration, or buffer exchange, using ultrafilters is a way forremoval and exchange of salts, sugars and the like. The person skilledin the art knows how to find the optimal conditions for eachpurification step. Also WO 98/22588, incorporated in its entirety byreference herein, describes methods for the production and purificationof adenoviral vectors. The methods comprise growing host cells,infecting the host cells with adenovirus, harvesting and lysing the hostcells, concentrating the crude lysate, exchanging the buffer of thecrude lysate, treating the lysate with nuclease, and further purifyingthe virus using chromatography.

Preferably, purification employs at least one chromatography step, asfor instance discussed in WO 98/22588, p. 61-70. Many processes havebeen described for the further purification of adenoviruses, whereinchromatography steps are included in the process. The person skilled inthe art will be aware of these processes, and can vary the exact way ofemploying chromatographic steps to optimize the process. It is forinstance possible to purify adenoviruses by anion exchangechromatography steps, see for instance WO 2005/080556 and Konz et al,2005, Hum Gene Ther 16: 1346-1353. Many other adenovirus purificationmethods have been described and are within the reach of the skilledperson. Further methods for producing and purifying adenoviruses aredisclosed in for example (WO 00/32754; WO 04/020971; U.S. Pat. Nos.5,837,520; 6,261,823; WO 2006/108707; Konz et al, 2008, Methods Mol Biol434: 13-23; Altaras et al, 2005, Adv Biochem Eng Biotechnol 99:193-260), all incorporated by reference herein.

For administering to humans, the invention may employ pharmaceuticalcompositions comprising the rAd and a pharmaceutically acceptablecarrier or excipient. In the present context, the term “Pharmaceuticallyacceptable” means that the carrier or excipient, at the dosages andconcentrations employed, will not cause any unwanted or harmful effectsin the subjects to which they are administered. Such pharmaceuticallyacceptable carriers and excipients are well known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed.,Mack Publishing Company [1990]; Pharmaceutical Formulation Developmentof Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition,A. Kibbe, Ed., Pharmaceutical Press [2000]). The purified rAd preferablyis formulated and administered as a sterile solution although it is alsopossible to utilize lyophilized preparations. Sterile solutions areprepared by sterile filtration or by other methods known per se in theart. The solutions are then lyophilized or filled into pharmaceuticaldosage containers. The pH of the solution generally is in the range ofpH 3.0 to 9.5, e.g pH 5.0 to 7.5. The rAd typically is in a solutionhaving a suitable pharmaceutically acceptable buffer, and the solutionof rAd may also contain a salt. Optionally stabilizing agent may bepresent, such as albumin. In certain embodiments, detergent is added. Incertain embodiments, rAd may be formulated into an injectablepreparation. These formulations contain effective amounts of rAd, areeither sterile liquid solutions, liquid suspensions or lyophilizedversions and optionally contain stabilizers or excipients. An adenovirusvaccine can also be aerosolized for intranasal administration (see e.g.WO 2009/117134).

For instance, adenovirus may be stored in the buffer that is also usedfor the Adenovirus World Standard (Hoganson et al, Development of astable adenoviral vector formulation, Bioprocessing March 2002, p.43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol. Another usefulformulation buffer suitable for administration to humans is 20 mM Tris,2 mM MgCl₂, 25 mM NaCl, sucrose 10% w/v, polysorbate-80 0.02% w/v.Obviously, many other buffers can be used, and several examples ofsuitable formulations for the storage and for pharmaceuticaladministration of purified (adeno)virus preparations can for instance befound in European patent no. 0853660, U.S. Pat. No. 6,225,289 and ininternational patent applications WO 99/41416, WO 99/12568, WO 00/29024,WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.

The invention is further explained in the following examples. Theexamples do not limit the invention in any way. They merely serve toclarify the invention.

EXAMPLES Example 1. Stabilizing the RSV F Protein in its Pre FusionConformation

Plasmids encoding basic RSV F sequences were synthesized and the aminoacid substitutions were introduced in the protein by site-directedmutagenesis. The protein variants were transiently expressed in HEK293cells. The relative protein expression on the cell surface was assessedby Flow Cytometry. The stability of the F proteins in pre-fusionconformation was evaluated in a heat-stability assay.

The protein sequence used for RSV A2 F protein variants was retrievedfrom the GenBank, accession number ACO83301.1. The amino acidsubstitutions were introduced in the sequence by site-directedmutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit, Agilenttechnologies). The mutagenesis primers were designed using on-line toolPrimerX. HEK293T cells (CRL-11268) were purchased from American TissueCulture Collection and cultured under standard cell culture conditions(37° C., 10% CO2).

Fully human IgG1 anti-RSV F protein antibodies CR9501 and CR9503 wereconstructed by cloning the heavy (VH) and light (VL) chain variableregions into a single IgG1 expression vector. PER.C6® cells (Crucell)were transfected with the IgG1 expression constructs and the expressedantibodies were purified from culture supernatants using POROSMabcapture A chromatography (Applied Biosystems) and then bufferexchanged to 50 mM NaAc, 50 mM NaCl, pH 5.5. Antibody concentration wasmeasured by optical absorption at 280 nm. Antibody quality was alsoconfirmed by size-exclusion chromatography (SEC), SDS-PAGE andisoelectric focusing. The antibody CR9501 comprises VH and VL regions of58C5 (as described in WO2011/020079) which binds specifically to RSV Fprotein in its pre-fusion conformation and not to the post-fusionconformation. CR9503 comprises VH and VL regions of motavizumab, whichrecognizes both the pre-fusion and post-fusion conformation of RSV F.

Protein Expression and Temperature Treatment:

The plasmids were transiently transfected into adherent HEK293T cellsusing 293fectine (Cat #12347-019) transfection reagents (LifeTechnologies) according to suppliers recommendations. 48 hours posttransfection the cells were harvested by detaching with EDTA-containingFACS buffer (no trypsin, see next section) and cell suspension washeat-treated for 10 minutes either in a water bath or in PCR block forthe temperature stability experiments. After the heat-treatment, thecells were prepared for the Flow Cytometry analysis.

For analysis of adeno expressed F proteins, A549 cells were infectedwith Ad26 virus at a MOI of 10 000 or 5000 and Ad35 viruses at a MOI of5000, 2500 or 1.250. After 48h, the cells were detached and heat treatedfor 15 minutes at 37° C., 50° C. and 56° C. Upon heat treatment cellswere stained using CR9501-Alexa647 or CR9503-Alexa647 and PropidiumIodide (PI). After staining, the cells were fixed and analyzed using theBD FACS CANTO II cell analyzer.

Flow Cytometry Analysis:

For each staining, the following controls were included: 1) negativecontrol sample. i.e. cells that were not subjected to any treatment andnot stained with any antibody labeled with a fluorophore; 2) positivecontrol samples, i.e. cells that are stained with only one fluorophore(one of each that are used for the experiment).

The cells were resuspended in the Flow Cytometry (FC Buffer, 5 mM EDTA,1% FBS in PBS) and distributed in volumes of 50 μl of the cellsuspension per well in a 96-well plate with a lid (U- or V-bottomplates). Two-step or one-step protocols were used for staining.

In case of the two-step protocol 50 μl of the first Abs (or buffer fornegative controls) was added to the wells and incubated at RT for 30min. Biotinylated CR9501 and CR9503 were used at 2 μg/ml (finalconcentration in a well 1 μg/ml). After incubation, the cells werewashed 2 times with the FC buffer. Afterwards 50 μl of Streptavidin-APC(Molecular Probes cat #SA1005, 0.1 mg/ml is used at 1:100) or buffer fornegative controls was added to the wells and incubated at RT for 30 min.The cells were washed again 2 times with the FC buffer. After the lastwash, the cells were resuspended in 100 μl of FC buffer+/−live-deadstain (PI from Invitrogen, cat #P1304MP, used at 2 μg/ml) and incubatedat RT for 15 minutes. The cells were centrifuged at 200 g (1000 rpm) for5 min., the buffer with PI was removed and the cells were resuspended in150 μl of the FC buffer.

In case of a one-step protocol, CR9501 and CR9503 antibodies werelabeled with fluorescent probe Alexa647 (Molecular Probes, cat #A-20186)according to manufacturer's instructions. Cells were stained accordingto the protocol above excluding the Streptavidin-APC step.

From the live cell population, the percentage of cells positive forCR9501/CR9503 antibody binding was determined. The cells positive forCR9503 binding express RSV F protein on their surface. The cellspositive for CR9501 binding express pre-fusion RSV F on their surface.

The intensity of the antibody staining (Median fluorescenceintensity—MFI) is proportional to the amount of F protein on the cellsurface. MFI was calculated from the live cell population expressing Fprotein.

Results:

Surface Cell Expression of the Full Length F Protein Variants:

A subset of mutations that was previously identified to increaseexpression or stability of the RSV F protein ectodomain in pre-fusionconformation was introduced in the wild type full length RSV A2 Fsequence (accession number Genbank ACO83301). The mutations wereintroduced alone or in multiple combinations, and the effect on proteinexpression and stability was assessed.

The expression level of the protein was measured as mean fluorescenceintensity (MFI) by Flow Cytometry after staining with the CR9503antibody that is recognizing both pre-fusion and post-fusion F protein.The combination of the two amino acid substitutions that were previouslydescribed for stabilization of the soluble RSV pre-F protein (i.e. N67Iand S215P) also increased the expression level of the full length RSV Fprotein by 2.3-fold, relative to wild type full length RSV F (FIG. 2 ).

A prominent increase in expression was observed for variants with 3amino acid substitutions combined. Interestingly, combination of morethan three mutations in one variant did not further increase proteinexpression. This may be due to limited capacity of the cellular membraneto accommodate multiple copies of F protein.

The amount of the pre-fusion F on the surface of the cell was assessedby staining with pre-fusion specific antibody CR9501 (FIG. 3 ).Transfection of the cells with all F variants resulted in a more or lesssimilar amount of pre-fusion F protein on the cell surface. Presence ofthe transmembrane domain stabilizes the full length protein to certainextent and therefore differences in the pre-fusion stability are not asapparent under ambient conditions between the full length F proteins.Therefore the heat-stability assay was developed to better discriminatestability of full length variants, as described below.

The A2 strain that was used as a parental sequence for the previouslydescribed F protein variants (WO2014/174018 and WO2014/202570) is a cellline adapted laboratory strain which has accumulated two unique and raremutations (i.e. of Lysine 66 and Isoleucine 76). In the presentinvention, these two residues were mutated to match the natural clinicalisolates (K66E, I76V). The K66E and I76V mutations were included inselected protein designs. In comparison to variants with Lys66 andIle76, variants with glutamate at 66 (K66E) have a tendency to expressslightly higher. Addition of valine at residue 76 (a double substitutionof K66E and I76V) does not influence expression level when compared tovariants with K66E substitution alone (FIG. 4 ).

Stability of the Full Length F Protein Variants on the Cell Surface:

In ambient conditions on a short time scale, no significant differencein stability of pre-fusion conformation was observed between full lengthF variants with the different combinations of stabilizing mutations. Anelevated temperature is known to serve as an efficient in vitro triggerfor refolding of RSV F protein from pre-fusion to post-fusionconformation. Therefore, a heat-shock assay was established and used toassess stability of the membrane-bound full length proteins. Shortly,the HEK293T cells were transfected with the F protein constructs andused for the assay 48 hours after transfection. The cells were detachedfrom cell culture dishes and the cell suspension was heat-treated atincreasing temperatures for 10 minutes. After the heat-treatment, cellswere stained with the anti-RSV F antibodies and analyzed by FlowCytometry. The Flow Cytometry data was analyzed in two different ways.The percentage of the cells, positive for staining with the anti-Fantibodies was analyzed, and also mean fluorescence intensity (MFI) ofthe positive cells was calculated (FIGS. 5A and 5B).

Both staining with CR9501 (antibody recognizing only pre-fusion Fprotein) and CR9503 (antibody recognizing both pre- and post-fusion Fprotein) were used in the Flow Cytometry assays. CR9503 antibody servedas a positive control. In case when F protein loses pre-fusionconformation but still is on the surface of the cell, the protein isstill detected with the CR9503 antibody. Loss of staining with bothantibodies indicates that protein is not available on the cell surfacefor antibody binding, e.g. due to aggregation.

Full length proteins with three of more amino acid substitutions weretested in the assay and compared to the wild type RSV F. The expressionof these variants was the highest and therefore these variants werepreferred candidates. All of the proteins contained the N67I and S215Psubstitutions, and one or two extra stabilizing mutations were added.

The unmodified wild type protein had a rather stable staining withCR9503 antibody. The MFI of the CR9503 staining was elevated at highertemperatures however the spread of values was also very high. Thisindicated that no protein aggregation occurred after the heat-shock.Half of the pre-fusion conformation was lost after incubation of cellsat approximately 55° C., after incubation of at 60° C. all pre-fusionconformation was lost as was demonstrated by decreased CR9501 binding tothe wild type F samples after heat-shock at increasing temperatures.

All tested pre-fusion F protein variants were more stable than the wildtype RSV F with majority of the CR9501 staining retaining also aftertreatment at higher temperatures (FIGS. 5A, 5B, and 6 ). Proteins withK498R amino acid substitution were less stable than the others. Additionof the K66E mutation further stabilized the proteins as also variantswith K498R amino acid substitution became as stable as others and noloss of the pre-fusion conformation was observed at 60° C. Only selectedcombinations of the stabilizing mutations were tested with K66E and I76Vcombined. All four tested proteins were stable when percentage ofpositive cells was analyzed, however when MFI was analyzed variant withK498R showed clear decrease in CR9501 binding after treatment with 60°C., indicating that this variant is less stable when evaluated in thetemperature stress assay.

In conclusion, a combination of three stabilizing mutations (includingN67I and S215P) was considered sufficient for high expression level andstability. The S46G or D486N mutations was selected as a thirdstabilizing mutation because of their position in the protein structure.K66E and I76V were included in the as they did not have negative effecton the protein expression and stability but made the sequence closer tonaturally occurring ones.

Thus, the pre-fusion RSV F protein with the mutations K66E, N67I, I76V,S215P and D486N (F2.2) (SEQ ID NO: 2) and the pre-fusion RSV F proteinwith the mutations K66E, N67I, I76V, S215P and S46G (F2.1) (SEQ IDNO: 1) were selected for the construction adenoviral vectors. Theseproteins were shown to be stable in the pre-fusion conformation in thetemperature stability assay up to 60° C., and to be expressed in highlevels.

Example 2. Preparation of Adenoviral Vectors

Cloning RSV F Gene into E1 Region of Ad35 and Ad26:

The nucleic acid sequences, coding for the pre-fusion F proteins of theinvention were gene optimized for human expression and synthesized, byGeneart. A Kozak sequence (5′ GCCACC 3′ (SEQ ID NO: 5)) was includeddirectly in front of the ATG start codon, and two stop codons (5′ TGATAA 3′ (SEQ ID NO: 6)) were added at the end of the RSV.pre-F codingsequence. The RSV.pre-F genes were inserted in the pAdApt35BSU plasmidand in the pAdApt26 plasmid via HindIII and XbaI sites.

Cell Culture:

PER.C6 cells (Fallaux et al., 1998, Hum Gene Ther 9: 1909-1917) weremaintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetalbovine serum (FBS), supplemented with 10 mM MgCl₂.

Adenovirus Generation, Infections and Passaging:

All adenoviruses were generated in PER.C6® cells by single homologousrecombination and produced as previously described (for rAd35: Havengaet al., 2006, J Gen. Virol. 87: 2135-2143; for rAd26: Abbink et al.,2007, J. Virol. 81: 4654-4663). Briefly, PER.C6 cells were transfectedwith Ad vector plasmids, using Lipofectamine according to theinstructions provided by the manufacturer (Life Technologies). Forrescue of e.g. Ad35 vectors carrying the RSV.pre-F transgenes expressioncassette, the pAdApt35BSU.RSV.pre-F plasmid andpWE/Ad35.pIX-rITR.dE3.5orf6 cosmid were used, whereas for Ad26 vectorscarrying the RSV.pre-F transgene expression cassette, thepAdApt26.RSV.pre-F plasmid and pWE.Ad26.dE3.5orf6.cosmid were used.Cells were harvested one day after full CPE, freeze-thawed, centrifugedfor 5 min at 3,000 rpm, and stored at −20° C. Next the viruses wereplaque purified and amplified in PER.C6 cultured on a single well of amultiwell 24 tissue culture plate. Further amplification was carried outin PER.C6 cultured using a T25 tissue culture flask and a T175 tissueculture flask. Of the T175 crude lysate, 3 to 5 ml was used to inoculate20×T175 triple-layer tissue culture flasks containing 70% confluentlayers of PER.C6 cells. The virus was purified using a two-step CsClpurification method. Finally, the virus was stored in aliquots at −85°C.

Example 3. Induction of Immunity Against RSV F Using RecombinantAdenovirus Serotypes 26 and 35 Expressing Pre Fusion RSV F In Vivo

The immunogenicity of Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 wasevaluated in mice, comparing cellular and humoral immune responses toresponses induced by identical doses of Ad26.RSV.FA2 (i.e. expressingthe wild type RSV F protein). Balb/c mice (n=4 per group) were immunizedwith the indicated dose of 10⁸ to 10¹⁰ viral particles (vp) Ad26.RSV.FA2or Ad26.RSV.preF2.1 or Ad26.RSV.preF2.2, or with formulation buffer. At8 weeks after prime, the number of RSV F A2 specific IFNγ spot formingunits (SFU) per 10⁶ splenocytes was determined using ELISpot. It wasshown that Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced increasedhumoral immune responses in mice when compared to Ad26.RSV.FA2, withbroad neutralizing capacity and maintained cellular responses. A singleintramuscular immunization with Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2elicited a cellular response (FIG. 7 ) which was characterized byinduction of CD8+ T cells positive for IFNγ, IL2 and/or TNFα (data notshown).

The quantity and quality of the cellular responses were comparablebetween Ad26.RSV.preF2.1, Ad26.RSV.preF.2.2 and Ad26.RSV.FA2. Incontrast, Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced significantlyhigher RSV neutralizing antibody titers than Ad26.RSV.FA2. Closeranalysis of the antibody responses demonstrated that Ad26.RSV.preF2.1and Ad26.RSV.preF.2.2 induced higher levels of antibodies againstpre-fusion F, while post-fusion F titers remained comparable toAd26.RSV.FA2, resulting in significantly increased preF/postF antibodyratios. In addition, the IgG2a/IgG1 ratio of the antibody responseremained unaltered, demonstrating a similar Th1 skewing of the humoralresponse as previously demonstrated for Ad26.RSV.FA2 (FIGS. 8A, 8B, and8C).

For Ad26.RSV.preF2.2 it was furthermore demonstrated that the antibodieselicited were capable of neutralizing various RSV A and B strains,laboratory strains as well as clinical isolates, similar as observed forAd26.RSV.FA2 (FIG. 9 ).

Subsequently, the efficacy and immunogenicity of Ad26.RSV.preF2.2 andAd35.RSV.preF2.2 vector constructs was evaluated in the cotton ratmodel. These animals are permissive to replication of human RSV, withpeak RSV titers in the lungs at days 4 and 5. Control groups in theexperiments included groups intranasally infected with a low dose RSVA2, thereby mimicking natural exposure, as well as groups immunized withFI-RSV, using the original lot 100 that induced enhanced respiratorydisease (ERD) in clinical studies in the dilution that was shown toinduce ERD in cotton rats.

Single intramuscular immunization of animals with Ad26.RSV.preF2.2 indoses ranging from 10⁵ to 10⁸ vp/animal, or Ad35.RSV.preF2.2 in dosesranging from 10⁶ to 10⁹ vp/animal resulted in complete protection of thelungs from infection with the vaccine homologous RSV A2 strain, exceptfor 3 animals immunized with 10⁵ vp Ad26.RSV.preF2.2 (FIGS. 10A and10B). Dose dependent protection of RSV replication in the nose wasobserved for both vectors. This ranged from full protection at 10⁸vp/animal, to partial protection at 10⁵ vp for Ad26.RSV.preF2.2, whereasfor Ad35.RSV.preF2.2, noses of animals immunized with 10⁹ vp were fullyprotected from RSV A2, and 10⁶ vp resulted in partial protection (FIGS.10C and 10D) Noses of animals immunized with Ad26.RSV.preF2.2 andAd35.RSV.preF2.2 were better protected from RSV A2 infection than whenimmunized with their respective wild type F counterparts Ad26.RSV.FA2and Ad35.RSV.FA2, when analyzed across dose (p=0.0003, and p=0.0001).Protection from RSV infection was accompanied by dose-dependentinduction of virus neutralization titers against RSV A Long, alreadyelicited by the lowest doses of Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2applied (FIGS. 10E and 10F). Across dose statistical comparisons of VNAA Long titers revealed that Ad26.RSV.preF2.2 is more immunogenic thanAd26.RSV.FA2 (p=0.0414), whereas elicitation of VNA titers was notsignificantly different between Ad35.RSV.preF2.2 and Ad35.RSV.FA2.

It was further demonstrated that Ad26.RSV.preF and Ad35.RSV.preF do notinduce histopathological signs of Enhanced Respiratory Disease (ERD)after RSV A2 challenge, at any of the concentrations tested. The cottonrat is the most used and best studied model to monitor ERD. In thisanimal model, vaccination with FI-RSV consistently induces ERD after RSVchallenge, which is visible by histopathological analysis of sections ofthe infected lungs for parameters as alveolitits, consisting primarilyof neutrophil infiltrates, and peribronchiolitis, consisting primarilyof lymphocyte infiltrates. In cotton rats, FI-RSV-induced scores forthese parameters can be observed from day 1 after RSV infection, andpeak at 4 to 5 days after RSV challenge.

ERD was analyzed 5 days after challenge with RSV A2 by scoring 4parameters of pulmonary inflammatory changes (peribronchiolitis,perivasculitis, interstitial pneumonia, alveolitis). Immunization withFI-RSV resulted in enhanced scores for most histopathological markers,which was especially apparent for alveolitis (FIG. 11 ), the marker thatwas previously shown to be the most discriminating marker for ERD. Noincreases in alveolitis or any other ERD histopathological marker wasobserved in animals immunized by either Ad26.RSV.preF2.2 orAd35.RSV.preF2.2 in a prime-only regimen after RSV challenge, even atlow vaccine doses that may induce low affinity and/or low levels ofantibodies (FIG. 11 ). This is confirming our previous results withAd26.RSV.FA2 and Ad35.RSV.FA2 vectors.

According to the invention, it has thus been shown that Ad26.RSV.preFand Ad35.RSV.preF are potent adenoviral vectors expressing RSV F A2which is stabilized in the pre-fusion conformation. These vectors inducestrong humoral and cellular immune responses. The immune responseelicited is protective against RSV A2 challenge and provides a widerange of virus neutralization in vitro against clinical and laboratoryisolates of RSV. No ERD induction was observed in cotton rats after RSVexposure of vaccinated animals and therefore confirms the data generatedwith Ad26 and Ad35 encoding for the wild type RSV F A2 antigen. Neithermice nor cotton rats showed overt signs of reactogenicity afterinjection of either Ad26.RSV.preF or Ad35.RSV.preF.

TABLE 1 Amino acid sequences of the RSV pre-fusion Fproteins encoded by the nucleic acid molecules ofthe invention (mutations are underlined)SEQ ID NO: 1: RSV preF2.1 amino acid sequence:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN SEQ ID NO: 2: RSV preF2.2 amino acid sequence:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

TABLE 2 Nucleotide sequence of preferred nucleic acidmolcules of the invention SEQ ID NO: 3: codon optimized nucleic acidencoding the RSV F pre-F2.1 pre-fusion protein PreF2.1ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAGTTCTACCAGAGCACCTGCAGCGCCGTGAGCAAGGGCTACCTGGGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGGAGATCAAGTGCAACGGCACCGACGCCAAGGTGAAGCTGATCAAGCAGGAGCTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACAGAGCCAGAAGAGAGCTGCCCAGATTCATGAACTACACCCTGAACAACGCCAAGAAGACCAACGTGACCCTGAGCAAGAAGAGAAAGAGAAGATTCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATCGCCAGCGGCGTGGCCGTGAGCAAGGTGCTGCACCTGGAGGGCGAGGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGAGCCTGAGCAACGGCGTGAGCGTGCTGACCAGCAAGGTGCTGGACCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACCGTGATCGAGTTCCAGCAGAAGAACAACAGACTGCTGGAGATCACCAGAGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGAGCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAGAAGCTGATGAGCAACAACGTGCAGATCGTGAGACAGCAGAGCTACAGCATCATGAGCATCATCAAGGAGGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAGGAGGGCAGCAACATCTGCCTGACCAGAACCGACAGAGGCTGGTACTGCGACAACGCCGGCAGCGTGAGCTTCTTCCCCCAGGCCGAGACCTGCAAGGTGCAGAGCAACAGAGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCAGCGAGGTGAACCTGTGCAACGTGGACATCTTCAACCCCAAGTACGACTGCAAGATCATGACCAGCAAGACCGACGTGAGCAGCAGCGTGATCACCAGCCTGGGCGCCATCGTGAGCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACAGAGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCGTGGACACCGTGAGCGTGGGCAACACCCTGTACTACGTGAACAAGCAGGAGGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAACGTGAACGCCGTGAAGAGCACCACCAACATCATGATCACCACCATCATCATCGTGATCATCGTGATCCTGCTGAGCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGAAGCACCCCCGTGACCCTGAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCCTTCAGCAACTGA SEQ ID NO: 4: codon optimized nucleic acidencoding the RSV F pre-F2.2 pre-fusion protein PreF2.2ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAGTTCTACCAGAGCACCTGCAGCGCCGTGAGCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGGAGATCAAGTGCAACGGCACCGACGCCAAGGTGAAGCTGATCAAGCAGGAGCTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACAGAGCCAGAAGAGAGCTGCCCAGATTCATGAACTACACCCTGAACAACGCCAAGAAGACCAACGTGACCCTGAGCAAGAAGAGAAAGAGAAGATTCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATCGCCAGCGGCGTGGCCGTGAGCAAGGTGCTGCACCTGGAGGGCGAGGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGAGCCTGAGCAACGGCGTGAGCGTGCTGACCAGCAAGGTGCTGGACCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACCGTGATCGAGTTCCAGCAGAAGAACAACAGACTGCTGGAGATCACCAGAGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGAGCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAGAAGCTGATGAGCAACAACGTGCAGATCGTGAGACAGCAGAGCTACAGCATCATGAGCATCATCAAGGAGGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAGGAGGGCAGCAACATCTGCCTGACCAGAACCGACAGAGGCTGGTACTGCGACAACGCCGGCAGCGTGAGCTTCTTCCCCCAGGCCGAGACCTGCAAGGTGCAGAGCAACAGAGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCAGCGAGGTGAACCTGTGCAACGTGGACATCTTCAACCCCAAGTACGACTGCAAGATCATGACCAGCAAGACCGACGTGAGCAGCAGCGTGATCACCAGCCTGGGCGCCATCGTGAGCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACAGAGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCGTGGACACCGTGAGCGTGGGCAACACCCTGTACTACGTGAACAAGCAGGAGGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCAACGAGTTCGACGCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAACGTGAACGCCGTGAAGAGCACCACCAACATCATGATCACCACCATCATCATCGTGATCATCGTGATCCTGCTGAGCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGAAGCACCCCCGTGACCCTGAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCCTTCAGCAACTGA

What is claimed is:
 1. A vaccine comprising a polynucleotide sequencethat encodes a respiratory syncytial virus (RSV) pre-fusion F proteinhaving the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.